ASSESSING BIOFILM ON BIOFILTER MEDIA (GRANULAR ACTIVATED CARBON AND ANTHRACITE) FROM A PILOT SCALE DRINKING WATER TREATMENT PLANT By Nick Dimas Bachelor of Science, Ryerson University, 2013 A thesis presented to Ryerson University in partial fulfillment of the requirements for the degree of Master of Applied Science in the Program of Environmental Applied Science and Management Toronto, Ontario, Canada, 2016 ©Nick Dimas, 2016 AUTHOR’S DECLARATION I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revision, as accepted by my examiners. I authorize Ryerson University to lend this thesis to other institutions or individuals for the purpose of scholarly research. I further authorize Ryerson University to reproduce this thesis by photocopying or by other means, in total or in part, at the request of other institutions or individuals for the purpose of scholarly research. I understand that my thesis may be made electronically available to the public. ii ASSESSING BIOFILM ON BIOFILTER MEDIA (GRANULAR ACTIVATED CARBON AND ANTHRACITE) FROM A PILOT SCALE DRINKING WATER TREATMENT PLANT Nick Dimas Master of Applied Science, Environmental Applied Science and Management, Ryerson University 2016 ABSTRACT Drinking Water Treatment Plants employ biofiltration systems to increase water quality through nutrient reduction. Microbial biofilms housed in biofilter media, are responsible for nutrient uptake and biodegradation. The purpose of this study was to re-evaluate the function and efficiency of biofilter media and investigate seasonal changes in the microbial populations. TOC and DO were more reduced in Granular Activated Carbon (GAC) media than in anthracite. Heterotrophic plate counts (HPC) were conducted to establish seasonal trends on microbial population. PCR-amplified 16S rRNA fragments were sequenced to compare microbial communities. Summer samples have higher HPC than winter samples. Summer samples yielded a reduction in microbial diversity and no detectable overlap with winter samples. Confocal microscopy conducted to qualitatively visualize the structure of biofilms was complemented by quantitative COMSTAT analysis showing GAC with double the biomass due to a greater level of attachment sites. GAC outcompetes anthracite in chemical adsorption and biological activity. iii Acknowledgements Over the last two and a half years this thesis has been the ongoing and primary focus of my life but would not have been possible without the combined efforts and support of a select few. First and foremost I would like to extend the deepest gratitude to my supervisors, Dr. Martina Hausner and Otini Kroukamp. You have both given me so much help and guidance for this study, making me very knowledgeable in microbiology and water treatment. Your dedication has inspired me to conquer challenges I faced and made me think critically. Thank you for all the time and effort you invested in me. You both have helped shape my love for academia and my inquisitiveness and thirst for knowledge. A special thanks to David Scott, operator of the pilot plant at the R.C. Harris Drinking Water Treatment Plant. Dave, you were an invaluable component to this study and have provided me with critical samples, information, and data that were essential for my research. You always made yourself available to me for any questions I needed answered, as well as continuously made me feel welcome to come back to the plant. Your hands on experience as well as your passion has made you an essential part of this team. I would also like to thank Dr. Gideon Wolfaardt who made the formal introduction between me and Dave, as well as meeting with me and help me think of how to establish the core elements of my project. In addition I would also like to thank Dr. Andrew Laursen who helped me so much with the statistical analysis of my data and use of ANOVA; this project’s statistical significance, is all thanks to him. Furthermore, I would like to thank Evan Ronan who has spent many hours training me with microscopy as well as teaching me how to use COMSTAT, many of these results were thanks to him. I would also like to thank all the members of the Wolfaardt/Gilbride Lab as well as the Haunser Lab who have been always been helpful and made working in the lab a pleasure. I would like to give special recognition and thank my family, but especially my parents, Who have continuously been there to support me no matter what. Their love, support, and encouragement gave me all the strength to carry on these past two and half years, and I could not have done it without them. Finally, I would like to thank my close friends who have been a constant source of motivation for me. You have all been there for me through the good times and the bad times. The laughter and jokes as well as advice has always kept me on track and no matter what happens I know, I can count on you guys for anything: Dragan Miscevic, Hirak Saxena, Paul Proios, Thierry Spiess, Brian Moroz, Tom Perivolaris, Theodore Nanos, and Devi Odisho. iv TABLE OF CONTENTS AUTHOR’S DECLARATION ......................................................................................................ii ABSTRACT ..................................................................................................................................iii ACKNOWLEDGEMENTS ......................................................................................................... iv LIST OF TABLES .......................................................................................................................vii LIST OF FIGURES ....................................................................................................................viii LIST OF ABBREVIATIONS ........................................................................................................x CHAPTER ONE: INRODUCTION............................................................................................1 1.1 WATER SCARCITY AND DRINKING WATER CONDITIONS....................................1 1.1.2 Excess Nutrients in Drinking Water..........................................................................2 1.2 REASONS/MOTIVATIONS............................................................................................5 1.3 EXPECTATIONS..............................................................................................................8 1.4 SCOPE OF WORK AND OBJECTIVES.......................................................................11 CHAPTER TWO: LITERATURE REVIEW.......................................................................14 2.1 TIMELINE OF BIOFILTRATION APPLICATIONS..................................................14 2.2 R.C. HARRIS WATER TREATMENT PROCESS......................................................15 2.2.1 The Pilot Scale.........................................................................................................16 2.3 BIOFILMS.......................................................................................................................19 2.4 BIOFILTER MEDIA......................................................................................................23 2.5 COMSTAT VARIABLE MEASUREMENTS………………………………………….29 CHAPTER THREE: METHODS AND MATERIALS......................................................32 3.1 SAMPLE RETRIEVAL..................................................................................................32 3.2 HETEROTROPHIC PLATE COUNTS.........................................................................33 3.3 DNA AMPLIFICATION AND SEQUENCING...........................................................33 3.4 CONFOCAL LASER SCANNING MICROSCOPY....................................................35 3.5 IMAGE ANALYSIS........................................................................................................36 3.6 STATISTICAL ANALYSIS……………………………………………………..…..37 CHAPTER FOUR: RESULTS................................................................................................38 4.1 HETEROTROPHIC PLATE COUNTS.........................................................................38 4.2 MICROBIAL DIVERSITY BASED ON CLASS.........................................................39 4.3 CONFOCAL LASER SCANNING MICROSCOPY....................................................41 4.4 IMAGE QUANTIFICATION.........................................................................................48 v 4.5 PILOT SCALE DOU AND TOCU DATA....................................................................53 CHAPTER FIVE: DISCUSSION.......................................................................................61 5.1 HETEROTROPHIC PLATE COUNTS.........................................................................62 5.2 MICROBIAL DIVERSITY.............................................................................................65 5.3 SEASONAL CHANGES.................................................................................................70 5.4 CLSM & IMAGE QUANTIFICATION OF BIOFILM PARAMETERS..................72 5.5 PILOT SCALE DOU AND TOCU DATA....................................................................81 5.6 ENHANCING BIOFILTERS.........................................................................................86 CHAPTER SIX: CONCLUSIONS AND FUTURE WORK.............................................89 APPENDIX 1.......................................................................................................................94